Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries.
Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries.
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通过基因重组 (ISOR) 掺入合成寡核苷酸：一种用于生成目标文库的多功能工具。
DOI：
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发表时间：
2007
期刊：
影响因子：
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通讯作者：
Dan S. Tawfik
中科院分区：
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作者：
Asael Herman;Dan S. Tawfik
蛋白质的定向进化极大地受益于位点特异性多样化方法，如饱和诱变。这些技术将多样性靶向蛋白质一级结构中通常不连续的许多选定位置。然而，靶向位置的数量可能很大，因此导致不切实际的大文库大小，其中几乎所有文库变体都是无活性的，并且选择期望性质的可能性极小。我们描述了一个通用的组合方法的部分多样化的大套残基。我们的文库寡核苷酸包含侧翼为野生型序列的随机密码子。将这些寡核苷酸添加到野生型基因片段的组装PCR中，将随机化的盒在其靶位点处并入重新组装的基因中。改变寡核苷酸浓度产生携带不同平均数目的突变位置的文库变体，所述突变位置包含整个多样化密码子组的随机子集。这种方法被称为通过基因重组（ISOR）来纯化合成寡核苷酸，用于创建胞嘧啶-C5甲基转移酶的文库，其中45个单独的位置被随机化。选择了一个文库，每个基因平均含有5.6个突变残基，并分离出具有野生型样活性的突变体。我们还创建了血清对氧磷酶PON 1库，其在活性位点周围的各个区域具有插入和缺失（indels）。筛选这些文库产生了一系列具有改变的底物特异性的突变体，并表明该酶的某些区域对indel具有令人惊讶的高耐受性。
The directed evolution of proteins has benefited greatly from site-specific methods of diversification such as saturation mutagenesis. These techniques target diversity to a number of chosen positions that are usually non-contiguous in the protein's primary structure. However, the number of targeted positions can be large, thus leading to impractically large library size, wherein almost all library variants are inactive and the likelihood of selecting desirable properties is extremely small. We describe a versatile combinatorial method for the partial diversification of large sets of residues. Our library oligonucleotides comprise randomized codons that are flanked by wild-type sequences. Adding these oligonucleotides to an assembly PCR of wild-type gene fragments incorporates the randomized cassettes, at their target sites, into the reassembled gene. Varying the oligonucleotides concentration resulted in library variants that carry a different average number of mutated positions that comprise a random subset of the entire set of diversified codons. This method, dubbed Incorporating Synthetic Oligos via Gene Reassembly (ISOR), was used to create libraries of a cytosine-C5 methyltransferase wherein 45 individual positions were randomized. One library, containing an average of 5.6 mutated residues per gene, was selected, and mutants with wild-type-like activities isolated. We also created libraries of serum paraoxonase PON1 harboring insertions and deletions (indels) in various areas surrounding the active site. Screening these libraries yielded a range of mutants with altered substrate specificities and indicated that certain regions of this enzyme have a surprisingly high tolerance to indels.